The Her2/neu tumor antigen, encoded by the erbB2/neu protooncogene, is a 185 kDa protein that belongs to the human epidermal growth factor receptor family. It consists of a cysteine-rich extracellular domain (ECD, from amino acids 23 to 652) with several glycosylation sites, a hydrophobic transmembrane domain (from amino acids 653 to 675) and an intracellular tyrosine kinase domain (from amino acids 676 to 1255). The Her2/neu receptor is expressed on the cell membrane of a variety of epithelial cell types and regulates aspects of cell growth and division through binding of specific growth factors. Her2/neu is expressed at low levels in many normal cells, but is over-expressed in a variety of cancers, including breast, ovarian, endometrial, gastric, pancreatic, prostate and salivary gland cancers.
For example, approximately 30% of metastatic breast cancers have been shown to over-express Her2/neu. This over expression is associated with a poor prognosis for the breast cancer patient, as it corresponds to decreased relapse-free periods and shortened survival time. Currently the most common forms of treating breast cancer involve surgery, chemical intervention and/or radiotherapy. Unless the cancer is restricted to a defined area, surgery alone cannot eliminate the cancer. Radiation treatment as well as chemotherapy may entail severe negative side effects.
In view of the disadvantages of the present therapies, attempts have been made to find additional approaches for treating e.g. breast cancer. One such approach is immunotherapy. One of the targets for an immunotherapeutic approach is the Her2/neu protein.
The clinical implications of Her2/neu over-expression in tumors have made Her2/neu an attractive target for antibody-mediated immunotherapy as an adjunct to conventional chemotherapy. However, the monoclonal antibody Trastuzumab (marketed as Herceptin®) is only effective in breast cancer where the Her2/neu receptor is over-expressed. Furthermore multiple infusions are required, resulting in high treatment costs.
Besides immunotherapy via passive immunization with monoclonal antibodies, further efforts have been focused on the active immunization and the identification of antigens recognized by human B- and T-lymphocytes. Such vaccine immunotherapy for cancer has been based on antigens against which humoral and/or cellular responses are elicited. These antigens should ideally be expressed or over expressed exclusively by the tumor and have been termed tumor-associated antigens (TAAs). One of the first TAAs described for breast cancer was HER2/neu. Meanwhile various TAAs representing different epitopes have been tested but so far none successfully passed product development.
In vaccinology, antigens intended to elicit an immune response are sometimes combined with one or more adjuvants, e.g. conjugated or otherwise associated with one or more delivery systems. Depending on the type of immune response intended (B or T cell response), different strategies are applied.
To induce a B cell (i.e. antibody) response the antigens should be B cell epitopes. As generally understood in the art, a B cell epitope is a part of an antigen that is recognized and bound by a B cell receptor. Lipids, polysaccharides and proteins/peptides may contain B cell epitopes which, upon introduction into an organism of choice, cause B cells to produce antibodies which specifically bind to the introduced epitope. The immunogenicity of some B cell epitopes can in some cases be increased by coupling to a suitable delivery system. Coupling of antigens intended to function as B cell epitopes to particles in a repetitive arrangement presumably enables cross-linking of the immunoglobulin receptors on the B cells, which is known to be an exceptionally strong activation signal. Repetitive arrangement can occur via fusion of the B cell epitope with a delivery system including e.g. hepatitis B core (HBc), keyhole limpet hemocyanins (KLH), tetanus toxoid (TT) and/or virosomes. For some B cell antigens T cell help can also enhance antibody production.
A promising approach of antitumor activity is based on the induction of tumor specific humoral immune responses; numerous antibodies directed against the extracellular domain (ECD) of Her2/neu have been generated by immunizing mice with cells expressing Her2/neu. The biological effect of these antibodies appears epitope-specific, that is it is based on specific recognition of a short subsequence within the Her2/neu ECD. Some antibodies have no effect or even actively stimulate tumor growth. The monoclonal antibody (mAb) 4D5 has been shown to reduce the growth of Her2/neu expressing tumors in mice by direct and indirect mechanisms such as apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Based on these results, a humanized form of this antibody, termed Trastuzumab (Herceptin®), was tested in clinical trials. Increased overall survival of patients with breast tumors overexpressing Her2/neu was observed following cytotoxic treatment plus Herceptin® as compared to chemotherapy or Trastuzumab alone. Herceptin® is now used as monotherapy but shows even higher efficacy in combination with cytotoxic chemotherapy.
Individual fragments of the ECD of Her2/neu are known in the art. For example, WO 2002/068474 (EP 1236740) relates to a vaccine that comprises a peptide of 9-25 amino acids which sequence occurs in the extracellular part of the Her2/neu protein. Further, WO 2007/118660 (EP 1884788) describes a multi-peptide vaccine comprising a specific combination of peptides presenting different amino acid sequences as occur in the extracellular part of the Her2/neu protein. These peptides in these publications may be administered individually or together, in the form of multiple discrete peptides, each preferably conjugated separately to a delivery system.
It is an aim of the present invention to provide improved substances suitable for use as the active components of a vaccine, as well as the corresponding vaccines themselves, for treating, preventing and/or ameliorating Her2-associated cancer. Ideally, such substances should improve upon the protective effect conferred by existing immunotherapies for such cancers while avoiding the need for laborious preparation techniques.